Developmentally regulated and cell type-specific expression of distinct sulfated glycosaminoglycan structures on cell surface proteoglycans is increasingly recognized as providing information relevant to cell-cell interactions and differentiation in developing organisms. In this report, developmental regulation of both the sulfation profile of chondroitin sulfate chains and activities of chondroitin 4-sulfotransferase (C4ST) and chondroitin 6-sulfotransferase (C6ST) were evaluated in embryonic chicken brain. The results revealed that the sulfation profile and the sulfotransferase activities changed markedly with development, and these alterations were precisely coordinated. Specifically, the proportions of both chondroitin 6-sulfate to 4-sulfate and C6ST to C4ST activities progressively decreased with development. In addition, the total amounts of both chondroitin sulfate chains and the sulfotransferase activities were highest during early embryonic stages and decreased sharply as the development reached completion. The developmental expression of the C6ST gene was also found to parallel the developmental down-regulation of both the C6ST activity and the chondroitin 6-sulfate structure. These findings suggest that the developmentally regulated expression of the sulfotransferases is a predominant factor for stage-specific regulation of chondroitin sulfate structures.

Among the many mammalian secreted phospholipase A 2 (sPLA 2 ) enzymes, PLA2G3 (group III secreted phospholipase A 2 ) is unique in that it possesses unusual N-and C-terminal domains and in that its central sPLA 2 domain is homologous to bee venom PLA 2 rather than to other mammalian sPLA 2 s. To elucidate the in vivo actions of this atypical sPLA 2 , we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA 2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA 2 s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5-or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A 2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.

The receptor for advanced glycation end-products (RAGE)-mediated cellular activation through the mitogen-activated protein kinase (MAPK) cascade, activation of NF-U UB and Rho family small G-proteins, cdc42/Rac, is implicated in the pathogenesis of in£ammatory disorders and tumor growth/metastasis. However, the precise molecular mechanisms for the initiation of cell signaling by RAGE remain to be elucidated. In this study, proteins which directly bind to the cytoplasmic C-terminus of RAGE were puri¢ed from rat lung extracts using an a⁄nity chromatography technique and identi¢ed to be extracellular signal-regulated protein kinase-1 and -2 (ERK-1/2). Their interactions were con¢rmed by immunoprecipitation of ERK-1/2 from RAGE-expressing HT1080 cell extracts with anti-RAGE antibody. Furthermore, the augmentation of kinase activity of RAGE-bound ERK upon the stimulation of cells with amphoterin was demonstrated by determining the phosphorylation level of myelin basic protein, an ERK substrate. In vitro binding studies using a series of C-terminal deletion mutants of human RAGE revealed the importance of the membrane-proximal cytoplasmic region of RAGE for the direct ERK^RAGE interaction. This region contained a sequence similar to the D-domain, a ERK docking site which is conserved in some ERK substrates including MAPK-interacting kinase-1/2, mitogen-and stress-activated protein kinase-1, and ribosomal S6 kinase. These data suggest that ERK may play a role in RAGE signaling through direct interaction with RAGE. ß

The relationship between sulfation and polymerization in chondroitin sulfate (CS) biosynthesis has been poorly understood. In this study, we investigated the specificity of bovine serum UDP-GalNAc: CS beta-GalNAc transferase responsible for chain elongation using structurally defined acceptor substrates. They consisted of tetra- and hexasaccharide-serines that were chemically synthesized and various regular oligosaccharides with a GlcA residue at the nonreducing terminus, prepared from chondroitin and CS using testicular hyaluronidase. The enzyme preparation was obtained from fetal bovine serum by means of heparin-Sepharose affinity chromatography. The preparation did not contain the alpha-GalNAc transferase recently demonstrated in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995), that utilizes common acceptor substrates. The beta-GalNAc transferase used as acceptors, two hexasaccharide-serines GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal (4-sulfate) beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, but neither the monosulfated hexasaccharide-serine GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser nor tetrasaccharide-serines with or without a sulfate group at C-4 of the third sugar residue Gal-3 from the reducing end. The results indicated that the sulfate group at the Gal-3 C-4 markedly affected the transfer of GalNAc to the terminal GlcA. In addition, a sulfate group at C-4 of the reducing terminal GalNAc of regular tetrasaccharides remarkably enhanced the GalNAc transfer, suggesting that the enzyme recognizes up to the fourth saccharide residue from the nonreducing end. The level of incorporation into a tetra- or hexasaccharide containing a terminal 2-O-sulfated GlcA residue was significant, whereas there was no apparent incorporation into tetra- or hexasaccharides containing a terminal 3-O-sulfated GlcA or penultimate 4,6-O-disulfated GalNAc residue. These results indicated that sulfation reactions play important roles in chain elongation and termination.

Hepatic fat deposition with hepatocellular damage, a feature of non-alcoholic fatty liver disease, is mediated by several putative factors including prostaglandins. In the present study, we examined whether group IVA phospholipase A2 (IVA-PLA2), which catalyzes the first step in prostanoid biosynthesis, is involved in the development of fatty liver, using IVA-PLA2-knockout mice. Male wild-type mice on high-fat diets (20% fat and 1.25% cholesterol) developed hepatocellular vacuolation and liver hypertrophy with an increase in the serum levels of liver damage marker aminotransferases when compared with wild-type mice fed normal diets. These high-fat diet-induced alterations were markedly decreased in IVA-PLA2-knockout mice. Hepatic triacylglycerol content was lower in IVA-PLA2-knockout mice than in wild-type mice under normal dietary conditions. Although high-fat diets increased hepatic triacylglycerol content in both genotypes, the degree was lower in IVA-PLA2-knockout mice than in wild-type mice. Under the high-fat dietary conditions, IVA-PLA2-knockout mice had lower epididymal fat pad weight and smaller adipocytes than wild-type mice. The serum level of prostaglandin E2, which has a fat storage effect, was lower in IVA-PLA2-knockout mice than in wild-type mice, irrespective of the kind of diet. In both genotypes, high-fat diets increased serum leptin levels equally between the two groups, but did not affect the serum levels of adiponectin, resistin, free fatty acid, triacylglycerol, glucose, or insulin. Our findings suggest that a deficiency of IVA-PLA2 alleviates fatty liver damage caused by high-fat diets, probably because of the lower generation of IVA-PLA2 metabolites, such as prostaglandin E2. IVA-PLA2 could be a promising therapeutic target for obesity-related diseases including non-alcoholic fatty liver disease.